Bone metastasis is the main cause of morbidity and mortality in prostate cancer (PCa) patients. Currently, PCa patients with bone metastatic disease respond poorly to chemotherapy. In cancer, deregulation of the receptor tyrosine kinase c-kit is a frequent event. However, its precise role in PCa and, more specifically in PCa bone metastasis, remains largely undetermined. In studies using clinical samples, we have shown that c-kit expression by prostate cells is gradually increased during malignant progression, with the highest expression seen in PCa bone metastases. We found that c-kit is neither expressed by PCa cell lines in vitro, nor by subcutaneous tumors formed by them. However, when these same cells are either co-cultured with bone marrow-derived cells or injected intraosseously using different experimental models, we found a de novo expression of c-kit in all the cases. Importantly, a correlation between c-kit expression and chemoresistance has been established in tumor systems other than prostate, and our preliminary data indicate that this might be also true for PCa, suggesting a role for c-kit in the poor response of patients with PCa bone metastasis to chemotherapy. Taken together, we hypothesize that de novo expression of c-kit induced by bone-derived factors is a prerequisite for the expansion of PCa cells within the bone, and is responsible for the acquisition of chemotherapy-resistance. To test our hypothesis, the specific aims are: (1) Determine the functional significance of bone-induced c-kit expression in PCa cells in intraosseous tumor growth and bone response, and the effect of de novo c-kit expression by PCa cells on the acquisition of chemoresistance, and (2) Identify causative bone-derived factors involved in the induction of c-kit in PCa cells. To provide causal evidence for the role of c-kit in intraosseous PCa growth, lentiviral-mediated RNA interference or transfect ion with a plasmid containing a dominant-negative (kinase-dead) c-kit mutant construct will be used to impede de novo c-kit induction in PCa cells within the bone microenvironment. The contribution of PCa-associated c-kit to intraosseous expansion of PCa cells will be studied using the SCID-hu model of bone metastasis developed in our laboratory, as well as its effect on osteoblast and osteoclast differentiations. The effect of de novo c-kit expression by PCa cells and its consequence to response to docetaxel will be studied in vivo and in vitro with c-kit-inducible and non-inducible c-kit PCa cells, and molecular mechanisms involved will be investigated. Using laser capture microdisection we will extract RNA from experimental PCa bone tumors and then use a) functional cloning from a retroviral cDNA expression library and b) differential expression analysis by gene microarray, to identify bone-derived factors inducing c-kit expression in PCa cells (Aim 2). It is expected that this high-risk, high-gain proposal will culminate in the design of new tailored therapies aimed at filling the gap in the treatment of PCa patients with bone metastasis. PUBLIC HEALTH RELEVANCE: Prostate cancer (PCa) is the most frequent and the second deadliest cancer affecting American men. As the cancer grows in the prostate, it can enter the bloodstream and spread (metastasize) to other parts of the body, usually into the bones. Unfortunately, the treatments currently available for prostate cancer patients with bone metastases are not curative. The objective of this proposal is to investigate the contribution of c-kit, a cell receptor, to metastasis of prostate cancer to the bone. The rationale behind this objective relates to our original findings in clinical and experimental samples that the expression of c-kit is augmented in PCa cells interacting with the bone microenvironment. Moreover, studies in cancers other than prostate and our preliminary results in PCa cells suggest that overexpression of c-kit in PCa cells renders them less susceptible to be killed by chemotherapy. Taken together, we hypothesize that de novo expression of c-kit induced by bone-derived factors is a prerequisite for the expansion of PCa cells within the bone, and is responsible for the acquisition of chemotherapy-resistance. To test our hypothesis, we will first modify PCa cells through genetic engineering, so that their cellular machinery will fail to produce c-kit. Injecting these cells directly within human bone fragments implanted in immunodepressed mice, and comparing the growth of these cells within bone with that of c-kit- inducible PCa, will allow us to determine how important the presence of c-kit is in PCa for their growth as bone metastasis. Then, using a similar experiment to that explained above, we will treat the mice injected with the c- kit-inducible and -non-inducible PCa cells with chemotherapy similar to that given to PCa patients. This study and others to be done in vitro will help us to confirm whether the production of c-kit by the cancer cells makes the cells less sensitive to killing by chemotherapy. Finally, we will microdissect areas immediately adjacent to prostate tumors growing within bone and analyze their genetic makeup with sophisticated techniques of molecular biology to identify factors present within the bone responsible for the induction of c-kit in the PCa cells. This finding could be of pivotal transcendence, as it will provide knowledge on how c-kit induction in PCa cells that metastasized to bone could be inhibited, thus avoiding its harmful consequences. This proposal is of high-impact because of the current lack of curative therapies for PCa bone metastasis. Understanding the function of c-kit in PCa cells is likely to shed light on a novel biological mechanism involved in bone metastasis, providing the foundation for the development of new tailored treatments towards eliminating morbidity and mortality due to this common complication in PCa patients.